1. Field of the Invention
The present invention relates generally to the fields of molecular biology, cell biology, pharmacology, developmental neuroscience, neurology, neurosurgery and regenerative biology. More particularly, it concerns methods and compositions for modulating regeneration of a nerve cell using a Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway. It also concerns methods and compositions for inhibiting growth of a neuron using inhibitors of neuronal growth that act via the Wnt signaling pathways, such as a Secreted Frizzled-Related Protein (sFRP), sFRP-like substance, Ryk, or Ryk-like substance.
2. Description of Related Art
The central nervous system (CNS) is connected by ascending sensory pathways and descending motor or regulatory pathways. In the CNS, somatosensory pathways ascend to the brain centers, and motor pathways controlling body movement descend from the brain to the spinal cord (Fitzgerald, 1996). The molecular mechanisms of axonal connections along the longitudinal axis of the CNS have remained a long-standing mystery.
Unlike the peripheral nervous system, damage to the central nervous system axons, such as spinal cord axons cannot be repaired, causing permanent impairment of neural function, such as in paralysis. The spinal cord serves important functions in the central nervous system. One such function is to allow communication of the body and the brain. The nerve fibers within the spinal cord carry messages to and from the brain to other parts of the body. In general sensory information from the body travels along the spinal cord up to the brain and instruction from the brain, such as motor command, travels along the spinal cord down from the brain. Thus, the spinal cord can be compared to a telephone cable, which connects the central office (brain) to the individual homes.
The term spinal cord injury refers to any injury of the neurons within the spinal canal. Spinal cord injury can occur from either trauma or disease to the vertebral column or the spinal cord itself. Most spinal cord injuries are the result of trauma to the vertebral column causing a fracture of the bone, or tearing of the ligaments with displacement of the bony column producing a pinching of the spinal cord. The majority of broken necks and broken backs, or vertebral fractures, do not cause any spinal cord damage; however, in 10-14% of the cases where a vertebral trauma has occurred, the damage is of such severity it results in damage to the spinal cord.
Spinal cord injury primarily occurs in young men with the greatest number of injuries occurring in the 16-30 age group. Patients with a spinal cord injury are designated as having tetraplegia (preferred to quadriplegia) or paraplegia. Tetraplegia refers to injuries to the cervical spinal cord and paraplegia refers to injuries below the cervical spinal cord. Patients with tetraplegia are slightly more common (51.7%) than patients with paraplegia. The majority of spinal cord injuries, about 37.4%, are sustained during a motor vehicle accident. Acts of violence are the second most common cause at 25.9%, falls are third at 21.5% and sports injuries are fourth at 7.1%.
It is estimated that the annual incidence of spinal cord injury (SCI), not including those who die at the scene of the accident, is approximately 40 cases per million population in the U.S., or approximately 11,000 new cases each year. The number of people in the U.S. who are alive today and who have SCI has been estimated to be between 721 and 906 per million population. This corresponds to between 183,000 and 230,000 persons.
Treatment options for patients with spinal cord injuries are limited. Often, patients with SCI are left with severe, permanent disabilities. A major reason for the limited availability of treatment options is the fact that there is little known about factors that can control and modulate nerve growth and regeneration following spinal cord injury. For example, the precise molecular mechanisms that guide axons along the anterior-posterior (A-P) axis of the spinal cord are unknown.
Axonal connections are patterned along the A-P and dorsal-ventral (D-V) neuraxes, wiring a large number of neurons into an intricate network. Axon guidance along the D-V axis has been a major focus of study in a number of experimental systems in recent years (Tessier-Lavigne and Goodman, 1996; Dickson, 2002). Much work has concentrated on the question of how axons are guided towards and away from the ventral midline and how midline crossing is regulated. Guidance molecules, such as Netrin-1 and members of the Slit and Semaphorin families, play pivotal roles in the dorsal-ventral guidance of axons (Tessier-Lavigne and Goodman, 1996; Dickson, 2002). The nature of the anterior-posterior guidance cues remains an enigma. Four classes of axon guidance molecules have been described (Tessier-Lavigne and Goodman, 1996): long-range attractants, long-range repellents, contact-mediated attractants and contact-mediated repellents. It is currently unknown whether a general gradient of attractant(s) or repellent(s) along the anterior-posterior axis guides axons to grow along this axis, or whether this guidance is mediated by more regional or segmental cues. The question of axon guidance along the A-P axis is of particularly interest in the spinal cord, where multiple classes of axons project either anteriorly or posteriorly along the length of the spinal cord. For example, somatosensory pathways ascend from the spinal cord to the brain and motor pathways descend from the brain to the spinal cord, with both the ascending and descending pathways carrying topographic information (FitzGerald, 1996).
The dorsal spinal cord commissural neurons form several ascending somatosensory pathways, such as the spinothalamic tracts, which send pain and temperature sensations to the brain (Ramon y Cajal, 1893; Altman and Bayer, 1984). The cell bodies of commissural neurons are located in the dorsal spinal cord. During embryonic development, commissural neurons project axons to the ventral midline. Once they reach the floor plate, they cross the midline and enter the contralateral side of the spinal cord. After midline crossing, commissural axons make a remarkably sharp anterior turn towards the brain (Ramon y Cajal, 1893; Altman and Bayer 1984; Tessier-Lavigne, 1994). All dorsal spinal cord commissural axons along the entire anterior-posterior length of the spinal cord project anteriorly after midline crossing. The initial ventral growth of the commissural axons is controlled by a gradient of a diffusible chemoattractant, Netrin-1 (Serafini et al., 1994; Kennedy et al., 1994; Serafini et al., 1996). As the axons cross the midline, they lose responsiveness to Netrin-1 (Shirasaki et al., 1998). Interestingly, while losing responsiveness to Netrin-1 during midline crossing, commissural axons gain responsiveness to several chemorepellents, which are located in the midline and the ventral spinal cord (Zou et al., 2000). These repellents help to expel the axons from the midline and to turn axons from their dorsal-ventral trajectory into their longitudinal pathways along the anterior-posterior axis by preventing axons from overshooting into the contralateral ventral spinal cord and recrossing the floor plate; the axons thus become “squeezed” into their longitudinal pathway (Zou et al., 2000). The expression pattern of the Slits and Semaphorins identified in these studies have been examined, but no anterior-posterior gradient of these chemorepellents in the spinal cord has been identified, suggesting that these repellents do not control anterior-posterior pathfinding.
Wnt polypeptides are secreted cysteine-rich glycosylated polypeptides that play a role in the development of a wide range of organisms. The Wnt family of polypeptides bind to an extracellular domain of a family of cell surface proteins called Frizzled receptors, and may play a role in embryonic induction, generation of cell polarity, and specification of cell fate.
Wnts are encoded by a large gene family, whose members have been found in round worms, insects, cartilaginous fish and vertebrates (Sidow, 1994). Wnts are thought to function in a variety of developmental and physiological processes since many diverse species have multiple conserved Wnt genes (McMahon, 1992; Nusse and Varmus, 1992). The Wnt growth factor family includes at least 18 genes identified in the human by cDNA cloning (see, e.g., Vant Veer et al., 1984; Miller, 2001).
Wnts may play a role in local cell signaling and neurogenesis. Biochemical studies have shown that much of the secreted Wnt protein can be found associated with the cell surface or extracellular matrix rather than freely diffusible in the medium (Papkoff and Schryver, 1990; Bradley and Brown, 1990). Studies of mutations in Wnt genes have indicated a role for Wnts in growth control and tissue patterning. In Drosophila, wingless (wg) encodes a Wnt gene (Rijsenijk et al., 1987) and wg mutations alter the pattern of embryonic ectoderm, neurogenesis, and imaginal disc outgrowth (Morata and Lawrence, 1977; Baker, 1988; Klingensmith and Nusse, 1994). Knock-out mutations in mice have shown Wnts to be essential for brain development (McMahon and Bradley, 1990; Thomas and Cappechi, 1990). However, a role for Wnts in mammalian directional axonal growth regulation in the spinal cord has not previously been suggested or considered.
The identification of modulators of neuronal growth and regeneration following SCI could be applied in new forms of treatment of patients with this debilitating condition. The identification of modulators of neuronal growth and regeneration could also be applied in the treatment of patients with other disorders involving neuronal dysfunction, such as neurodegenerative diseases. Agents that can promote axonal growth along the A-P axis following injury to the spinal cord may be applied to help prevent the permanent paralysis that is often associated with SCI. Therefore, there is a need for better treatments of SCI, and a greater understanding of modulators of neuronal growth and regeneration might lead to improved methods of treatment of this devastating disorder.